These results show that proton minibeam radiation therapy results in reduced adverse effects compared with conventional homogeneous broad-beam irradiation and, therefore, might have the potential to decrease the incidence of side effects resulting from clinical proton and/or heavy ion therapy.

The application of a microchannel proton irradiation was compared to homogeneous irradiation in a three-dimensional human skin model. The goal is to minimize the risk of normal tissue damage by microchannel irradiation, while preserving local tumor control through a homogeneous irradiation of the tumor that is achieved because of beam widening with increasing track length. 20 MeV protons were administered to the skin models in 10- or 50-μm-wide irradiation channels on a quadratic raster with distances of 500 μm between each channel (center to center) applying an average dose of 2 Gy. For comparison, other samples were irradiated homogeneously at the same average dose. Normal tissue viability was significantly enhanced after microchannel proton irradiation compared to homogeneous irradiation. Levels of inflammatory parameters, such as Interleukin-6, TGF-Beta, and Pro-MMP1, were significantly lower in the supernatant of the human skin tissue after microchannel irradiation than after homogeneous irradiation. The genetic damage as determined by the measurement of micronuclei in keratinocytes also differed significantly. This difference was quantified via dose modification factors (DMF) describing the effect of each irradiation mode relative to homogeneous X-ray irradiation, so that the DMF of 1.21 ± 0.20 after homogeneous proton irradiation was reduced to 0.23 ± 0.11 and 0.40 ± 0.12 after microchannel irradiation using 10- and 50-μm-wide channels, respectively. Our data indicate that proton microchannel irradiation maintains cell viability while significantly reducing inflammatory responses and genetic damage compared to homogeneous irradiation, and thus might improve protection of normal tissue after irradiation.

The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened.Ionizing radiation induces a variety of different types of damage when targeted to living cells. Severe damage, which can influence cell survival or lead to carcinogenesis, occurs due to ionizing events in the DNA molecule itself. The most lethal of these types of DNA damages are the double-strand breaks (DSB), as they may lead to genetic alterations which in turn can be responsible for cell death or carcigonesis. Mammalian cells react with a variety of complex response mechanisms to DSB induction. One main reaction is the phosphorylation of the histone variant H2AX at serine 139 (S139) to obtain γ H2AX through kinases such as ATM, ATR and DNA-PK 1 . The γ H2AX domains occur in mega-base-pair (Mbp) large regions of the chromatin around DSB 2-6 and can be visualized as so-called ionizing radiation induced foci (IRIF) 7 . The recruitment and activation of proteins due to damage induction can later on lead to the repair of DSB. The cell has different repair mechanisms to properly rejoin the ends of a DSB, including the possibly error-prone non-homologous end joining (NHEJ) 8 and the in most cases error-free homologous recombination (HR) 9 . HR is limited to the S/G2 cell cycle phase, due to the fact that a homologous sister chromatin is needed in close vicinity to the DSB as a template to repair the damaged chromatin 2-4 . As a backup pathway for failed NHEJ in G1 an alternative end-joining pathway (alt-EJ) has previously been identified, which works as a last resort, when the other pathways fail 8 . Recent work analyzed the clustering of DSB repair factors in detail using high resolution microscopy 10-12 and nanoscopy 11,[13][14][15][16] in combination with state of the art correlation and clustering analysis methods. With these methods it is possible to gain a deeper understanding of the functionality of DSB repair proteins and their interactions. After the first reactions to DSB induction, such as phosphorylation of H2AX (γ H2AX), the recruitment of downstream repair proteins starts for NHEJ as well as f...